Shy Matilda Hunsdorfer, nicknamed Tillie, prepares an experiment involving marigolds raised from seeds exposed to radioactivity for her science fair. She is, however, constantly thwarted by her mother Beatrice, who is self-centered and abusive, and by her extroverted and unstable sister Ruth, who submits to her mother's will. Over the course of the play, Beatrice constantly tries to stamp out any opportunities Tillie has of succeeding, due to her own lack of success in life. As the play progresses, the paths of the three characters diverge: Tillie wins the science fair through perseverance; Ruth attempts to stand up to her mother but has a nervous collapse at the end of the play, and Beatrice—driven to the verge of insanity by her deep-seated enmity towards everyone—kills the girls' pet rabbit Peter and ends up wallowing in her own perceived insignificance. Despite this, Tillie (who is much like her project's deformed but beautiful and hardy marigolds) secretly continues to believe that everyone is valuable.[citation needed]

The main protagonist of the play. A quiet and introverted character who is teased at school. She copes with her life by immersing herself in science, hoping to reach a philosophical epiphany. Her untiring quest for her individuality stands in open defiance of her mother's wish for total control over the family. Consequently, she receives the brunt of the abuse. Tillie also owns a rabbit named Peter, given to her by her science teacher, Mr. Goodman.

Ruth Hunsdorfer

Tillie's older sister. A brash but confused adolescent, she looks to others for advice, but often gains this insight from Beatrice. Although abused as well, she often bends to her mother's will, putting her in her mother's favor and sheltering her from the full extent of her mother's abuse. On many occasions, she attempts to make Tillie look like the crazy one, even though she herself underwent psychiatric treatment. She also takes a liking to Tillie's pet rabbit, to the point where she blackmails Tillie for possession of the rabbit, and threatens to tell her mother what the adults called mother at the school (Betty the Loon). There is evidence in the story suggesting that Ruth may have epilepsy.

Tillie's science teacher. He serves as a mentor to Tillie. Mr. Goodman is mentioned on many occasions, but never seen, although the dialogue often implies that he is the only positive role model in Tillie's life.

Nanny

An elderly boarder in the Hunsdorfer household. Silent throughout, she doesn't contribute much beyond being yet another burden to the already stressed-out Beatrice who verbally abuses her as she does her daughters. Veteran actress Judith Lowry played "Nanny" in nearly every production of the play, including TV, Off-Broadway and the movie.[6] During the off-Broadway run, even though her character never spoke any lines in the play, she became a popular guest on the Tonight Show with Johnny Carson and that led to late stardom as a series regular on Phyllis with Cloris Leachman.[7]

Mr Frank

Beatrice's father. A deceased vegetable vendor. After his wife (Beatrice's mother) died, he raised Beatrice on his own. Although he had a lowly status, Beatrice holds him in high regard—"He makes up for all the men in the world"—and smiles imagining her daughters meeting him.

Janice Vickery

Tillie's rival at the science fair. Her experiment involved boiling the skin off a dead cat so she may use its skeleton. She plans to use a dog in her next science fair project.